DOCSLIB.ORG

Exogenous PPAR 2 but Not PPAR 1 Reactivates Adipogenesis

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Exogenous PPAR 2 but Not PPAR 1 Reactivates Adipogenesis Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION ␥ differential isoform function. Rationally engineered PPAR knockdown by transcription factors potentially provide a powerful tool engineered transcription for targeted regulation of endogenous genes by combin- ing a functional transcription regulatory domain with a factors: exogenous PPAR␥2 customized DNA binding domain that can bind to a spe- but not PPAR␥1 cific sequence within the target gene. C2H2 zinc finger proteins (ZFPs) can be engineered to bind with high reactivates adipogenesis specificity to wide a diversity of DNA sequences (Des- jarlais and Berg 1992; Choo and Klug 1994; Jamieson et Delin Ren,1 Trevor N. Collingwood,2 al. 1994; Rebar and Pabo 1994; Greisman and Pabo 1997). Edward J. Rebar,2 Alan P. Wolffe,2 Previous studies have demonstrated the utility of both and Heidi S. Camp1,3 engineered activator- and repressor-ZFPs in the regula- tion of endogenous chromosomal loci (Bartsevich and 1Department of Molecular Science and Technology, Pfizer Juliano 2000; Beerli et al. 2000; Zhang et al. 2000; Liu et Global and Research Development, Ann Arbor, Michigan al. 2001). Our goal for this study was to selectively in- 48105, USA; 2Sangamo BioSciences Inc., Pt. Richmond, hibit expression of the PPAR␥2 isoform in the adipo- California 94804, USA genic mouse 3T3-L1 cell line by utilizing engineered zinc finger repressor proteins. To determine functional differences between the two splice variants of PPAR␥ (␥1 and ␥2), we sought to se- Results and Discussion ␥ lectively repress 2 expression by targeting engineered The mouse PPAR␥ gene spans >105 kb (Zhu et al. 1995). ␥ zinc finger repressor proteins (ZFPs) to the 2-specific Coding exons 1 to 6 are conserved between the ␥1 and ␥2 promoter, P2. In 3T3-L1 cells, expression of ZFP55 re- isoforms (Fig. 1A) and transcription of these is driven by sulted in >50% reduction in ␥2 expression but had no an upstream promoter (P1) that also drives expression of effect on ␥1, whereas adipogenesis was similarly reduced two untranslated ␥1-specific exons, A1 and A2. The ad- by 50%. However, ZFP54 virtually abolished both ␥2 ditional amino acids at the amino terminus of ␥2 are and ␥1 expression, and completely blocked adipogenesis. encoded by an additional exon, B1, that is uniquely regu- Overexpression of exogenous ␥2 in the ZFP54-expressing lated by a separate promoter (P2) lies >63 kb downstream ␥ cells completely restored adipogenesis, whereas overex- of P1. DNase I digestion of the endogenous PPAR gene locus in 3T3-L1 cells revealed two DNase I hypersensi- pression of ␥1 had no effect. This finding clearly identi- ␥ tive sites in the vicinity of the proximal P2 promoter fies a unique role for the PPAR 2 isoform. that represent regions of accessible chromatin at an en- Received October 12, 2001; revised version accepted dogenous locus (Fig. 1B; DHS1 and DHS2). Two six-fin- November 15, 2001. ger ZFPs (ZFP54 and ZFP55; Fig. 1C) linked to the KRAB transcriptional repression domain (Margolin et al. 1994) were designed to bind specifically to 18-bp sequences The nuclear hormone receptor PPAR␥ is essential for within the DHS1 shown in Figure 1B. Each ZFP bound cellular differentiation and lipid accumulation during its cognate site on naked DNA with high affinity adipogenesis (Barak et al. 1999; Kubota et al. 1999; Rosen (Kd = 20 and 44 pM, respectively). et al. 1999). The adipocyte-specific ␥2 isoform differs The ZFP54 and ZFP55 repressor proteins were ex- from the more widely expressed ␥1 in that it contains pressed retrovirally in 3T3-L1 cells to similar levels (Fig. additionally 30 amino acid residues at the amino termi- 2A). Nontransduced wild-type cells and stable pools of nus (Kliewer et al. 1994; Tontonoz et al. 1994a; Zhu et al. infected cells expressing each ZFP, as well as control 1995). Evidence suggests these residues contribute to a cells retrovirally transduced to express LacZ, were in- constitutive transcription activation function that is duced to differentiate and initiate the adipogenic path- 5–10-fold greater than in ␥1 (Werman et al. 1997). way. Total PPAR␥ mRNA level was determined at 0, 2, PPAR␥2 is selectively expressed in adipose tissue (Fajas and 5 d post induction. Over the 5-d time course PPAR␥ et al. 1997) and is strongly up-regulated during adipogen- expression increased ∼5.8- to 6.0-fold in both the wild- esis (Tontonoz et al. 1994b; Wu et al. 1998), suggesting a type and LacZ control cells (Fig. 2B). Up-regulation of specific role for this isoform in fat cell differentiation. total PPAR␥ expression was reduced slightly in the pres- Nevertheless, a specific role for ␥2 that could not be ence of ZFP55 (4.8-fold compared to 6.0-fold in wild type) substituted by ␥1 has not been clearly determined. but completely inhibited by ZFP54. However, a time The ability to selectively knock out or knock down course analysis of expression of the individual PPAR␥ the expression of a specific gene provides a powerful ap- isoforms revealed that although ZFP54 effectively inhib- proach for understanding its biological function. The tar- ited expression of both isoforms, ZFP55 selectively re- geting of individual mRNA splice variants offers an even pressed PPAR␥2by∼50% and had little effect on greater level of selective control and understanding of PPAR␥1 compared with wild-type cells (Fig. 2C). An- other ZFP–KRAB fusion protein that binds to a sequence that is absent in the PPAR␥ gene has no effect on PPAR␥ [Key Words: PPAR␥1; PPAR␥2; adipogenesis; zinc finger protein] expression or adipogenesis (data not shown). The effects 3 Corresponding author. of these ZFPs are confirmed at the protein level whereby E-MAIL [email protected]; FAX (734) 622-5668. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ PPAR␥2 expression is virtually knocked out in the pres- gad.953802. ence of ZFP54, whereas only a low but detectable level of GENES & DEVELOPMENT 16:27–32 © 2002 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/02 $5.00; www.genesdev.org 27 Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Ren et al. Figure 1. Structure of PPAR␥ gene and location of accessible chromatin in P2 promoter. (A) Genomic structure of mPPAR␥ showing exon splicing (Zhu et al. 1995). (B) Location of DNase I hypersensitive sites (DHS1 and DHS2) in the proximal P2 pro- moter, along with positions of ZFPs binding sites. (C) ZFP target sequences and finger designs. (Target sequence) The promoter DNA sequence to which each ZFP was designed. (Finger de- signs) The residues in each position from −1 to +6 of the recog- nition helix of each finger targeted against the cognate basepair ␥ triplet subsite. Figure 2. Ectopic expression of PPAR 2-specific ZFPs in 3T3- L1 cells. 3T3-L1 cells were infected with retroviral vectors ex- pressing PPAR␥2 promoter-specific zinc finger repressors, PPAR␥1 remains (Fig. 2D). In the presence of ZFP55 only ZFP54 and ZFP55. The uninfected 3T3-L1 cells (wild type) or PPAR␥2 expression is reduced substantially by ZFP55. cells infected with retroviral vector expressing LacZ gene were Because P1 and P2 are >63 kb apart, it was predicted that used as negative controls. (A) Total RNA was isolated from ZFPs targeted to P2 would selectively inhibit PPAR␥2 selected cell lines and determined the levels of ZFP mRNA by expression. This was the case with ZFP55, which gave Northern blot analysis using a 300 bp C-terminal fragment of 50% knockdown of PPAR␥2 only, however ZFP54 sup- ZFP as probe. The equivalence of RNA loading was verified by pressed both isoforms almost completely. ZFP54 and ␤-actin. (B) Total RNA was isolated on indicated days postdif- ZFP55 bind to opposite strands of the DNA and have ferentiation and subjected to RNase protection assay using a slightly different DNA binding affinities, which may PPAR␥-specific riboprobe. Arrows indicate the protected contribute to their differential effects on P2. PPAR␥ and ␤-actin mRNAs. The fold-induction was calculated The promoter specificity exhibited by the two ZFPs by normalization of PPAR␥-specific signal with ␤-actin signal facilitates examination of the functional requirements and presented as fold-change compared to day-0 of wild-type for each PPAR␥ isoform in adipogenesis. In complete cells. (C) The real time quantitative RT–PCR (TaqMan) analysis concordance with inhibited mRNA and protein expres- of PPAR␥1 and PPAR␥2 mRNA expression. 40 ng and 60 ng of sion, we find that ZFP54 totally blocks the cellular lipid reversed transcribed total RNA was used for PPAR␥2 and accumulation that is a marker of adipogenesis (Fig. 3). PPAR␥1 analysis, respectively. (D) Western blot analysis with a Furthermore, the cells in which only PPAR␥2 is selec- polyclonal antibody against PPAR␥. The arrows indicate tively repressed by 50% (ZFP55) also show a correspond- PPAR␥1 and PPAR␥2 proteins. 28 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Functional differences between PPAR␥1 and PPAR␥2 also was not inhibited, but rather an increase in its ex- pression was observed in the presence of ZFP54. This suggests a potential negative feedback mechanism whereby PPAR␥ may actively down-regulate C/EBP␦,in keeping with the reduction in C/EBP␦ observed during progression of adipogenesis (Wu et al. 1998). Taken to- gether these results support the notion that the inhibi- tory effects of ZFPs are specific. Genes that are expressed during adipogenesis after PPAR␥ induction include C/EBP␣ and the adipocyte-spe- cific aP2 (Wu et al.
Load more

Recommended publications
  • Engineering Zinc-Finger Protein and CRISPR/Cas9 Constructs to Model the Epigenetic and Transcriptional Phenomena That Underlie Cocaine-Related Behaviors
    Engineering zinc-finger protein and CRISPR/Cas9 constructs to model the epigenetic and transcriptional phenomena that underlie cocaine-related behaviors Hamilton PJ, Heller EA, Ortiz Torres I, Burek DD, Lombroso SI, Pirpinias ST, Neve RL, Nestler EJ Multiple studies have implicated genome-wide epigenetic remodeling events in brain reward regions following drug exposure. However, only recently has it become possible to target a given type of epigenetic remodeling to a single gene of interest, in order to probe the causal relationship between such regulation and neuropsychiatric disease (Heller et al., Nat Neurosci, 2014). Our group has successfully utilized synthetic zinc- finger proteins (ZFPs), fused to either the transcriptional repressor, G9a, that promotes histone methylation or the transcriptional activator, p65, that promotes histone acetylation, to determine the behavioral effects of targeted in vivo epigenetic reprogramming in a locus-specific and cell-type specific manner. Given the success of our ZFP approaches, we have broadened our technical repertoire to include the more novel and flexible CRISPR/Cas9 technology. We designed guide RNAs to target nuclease-dead Cas9 (dCas9) fused to effector domains to the fosB gene locus, a locus heavily implicated in the pathogenesis of drug abuse. We observe that dCas9 fused to the transcriptional activator, VP64, or the transcriptional repressor, KRAB, and targeted to specific sites in the fosB promoter is sufficient to regulate FosB and ΔFosB mRNA levels, in both cultured cells and in the nucleus accumbens (NAc) of mice receiving viral delivery of CRISPR constructs. Next, we designed a fusion construct linking the dCas9 moiety to a pseudo-phosphorylated isoform of the transcription factor CREB (dCas9­ CREB(S133D)).
    [Show full text]
  • Comprehensive Genome-Wide Exploration of C2H2 Zinc Finger Family in Grapevine (Vitis Vinifera L.): Insights Into the Roles in the Pollen Development Regulation
    G C A T T A C G G C A T genes Article Comprehensive Genome-Wide Exploration of C2H2 Zinc Finger Family in Grapevine (Vitis vinifera L.): Insights into the Roles in the Pollen Development Regulation Oscar Arrey-Salas 1,* , José Carlos Caris-Maldonado 2, Bairon Hernández-Rojas 3 and Enrique Gonzalez 1 1 Laboratorio de Genómica Funcional, Instituto de Ciencias Biológicas, Universidad de Talca, 3460000 Talca, Chile; [email protected] 2 Center for Research and Innovation (CRI), Viña Concha y Toro, Ruta k-650 km 10, 3550000 Pencahue, Chile; [email protected] 3 Ph.D Program in Sciences Mention in Modeling of Chemical and Biological Systems, Faculty of Engineering, University of Talca, Calle 1 Poniente, 1141, 3462227 Talca, Chile; [email protected] * Correspondence: [email protected] Abstract: Some C2H2 zinc-finger proteins (ZFP) transcription factors are involved in the development of pollen in plants. In grapevine (Vitis vinifera L.), it has been suggested that abnormalities in pollen development lead to the phenomenon called parthenocarpy that occurs in some varieties of this cultivar. At present, a network involving several transcription factors types has been revealed and key roles have been assigned to members of the C2H2 zinc-finger proteins (ZFP) family in model plants. However, particularities of the regulatory mechanisms controlling pollen formation in grapevine Citation: Arrey-Salas, O.; remain unknown. In order to gain insight into the participation of ZFPs in grapevine gametophyte Caris-Maldonado, J.C.; development, we performed a genome-wide identification and characterization of genes encoding Hernández-Rojas, B.; Gonzalez, E. ZFP (VviZFP family).
    [Show full text]
  • A New C2H 2 Zinc Finger Protein and a C2C2 Steroid Receptor-Like Component
    Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Proteins that bind to Drosophila chorion cis-regulatory elements: A new C2H 2 zinc finger protein and a C2C2 steroid receptor-like component Martin J. Shea, 1 Dennis L. King, 1 Michael J. Conboy, 2 Brian D. Mariani, 3 and Fotis C. Kafatos 1,3 ~Department of Cellular and Developmental Biology, Harvard University Biological Laboratories, Cambridge, Massachusetts 02138 USA; ZJefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 USA; 3Institute of Molecular Biology and Biotechnology, Research Center of Crete, and Department of Biology, University of Crete, Heraklion 711 10, Crete, Greece Gel mobility-shift assays have been used to identify proteins that bind specifically to the promoter region of the Drosophila s15 chorion gene. These proteins are present in nuclear extracts of ovarian follicles, the tissue where s15 is expressed during development, and bind to specific elements of the promoter that have been shown by transformation analysis to be important for in vivo expression. The DNA binding specificity has been used for molecular cloning of two components from expression cDNA libraries and for their tentative identification with specific DNA-binding proteins of the nuclear extracts. The mRNAs for both of these components, CF1 and CF2, are differentially enriched in the follicles. DNA sequence analysis suggests that both CF1 and CF2 are novel Drosophila transcription factors. CF2 is a member of the C2H2 family of zinc finger proteins, whereas CF1 is a member of the family of steroid hormone receptors. The putative DNA-binding domain of CF1 is highly similar to the corresponding domains of certain vertebrate hormone receptors and recognizes a region of DNA with similar, hyphenated palindromic sequences.
    [Show full text]
  • Zinc Finger Proteins: Getting a Grip On
    Zinc finger proteins: getting a grip on RNA Raymond S Brown C2H2 (Cys-Cys-His-His motif) zinc finger proteins are members others, such as hZFP100 (C2H2) [11] and tristetraprolin of a large superfamily of nucleic-acid-binding proteins in TTP (CCCH) [12], are involved in histone pre-mRNA eukaryotes. On the basis of NMR and X-ray structures, we processing and the degradation of tumor necrosis factor know that DNA sequence recognition involves a short a helix a mRNA, respectively. In addition, there are reports of bound to the major groove. Exactly how some zinc finger dual RNA/DNA-binding proteins, such as the thyroid proteins bind to double-stranded RNA has been a complete hormone receptor (CCCC) [13] and the trypanosome mystery for over two decades. This has been resolved by the poly-zinc finger PZFP1 pre-mRNA processing protein long-awaited crystal structure of part of the TFIIIA–5S RNA (CCHC) [14]. Whether their interactions with RNA are complex. A comparison can be made with identical fingers in a based on the same mechanisms as protein–DNA binding TFIIIA–DNA structure. Additionally, the NMR structure of is an intriguing structural question that has remained TIS11d bound to an AU-rich element reveals the molecular unanswered until now. details of the interaction between CCCH fingers and single-stranded RNA. Together, these results contrast the What follows is an attempt to expose both similarities different ways that zinc finger proteins bind with high and differences between C2H2 zinc finger protein bind- specificity to their RNA targets. ing to RNA and DNA based on recent X-ray structures.
    [Show full text]
  • PARP13 Regulates Cellular Mrna Post-Transcriptionally and Functions As a Pro-Apoptotic Factor by Destabilizing TRAILR4 Transcript
    ARTICLE Received 4 Sep 2014 | Accepted 23 Sep 2014 | Published 10 Nov 2014 DOI: 10.1038/ncomms6362 PARP13 regulates cellular mRNA post-transcriptionally and functions as a pro-apoptotic factor by destabilizing TRAILR4 transcript Tanya Todorova1,2, Florian J. Bock2 & Paul Chang1,2 Poly(ADP-ribose) polymerase-13 (PARP13/ZAP/ZC3HAV1) is an antiviral factor, active against specific RNA viruses such as murine leukaemia virus, Sindbis virus and human immunodeficiency virus. During infection, PARP13 binds viral RNA via its four CCCH-type zinc-finger domains and targets it for degradation by recruiting cellular messenger RNA (mRNA) decay factors such as the exosome complex and XRN1. Here we show that PARP13 binds to and regulates cellular mRNAs in the absence of viral infection. Knockdown of PARP13 results in the misregulation of hundreds of transcripts. Among the most upregulated transcripts is TRAILR4 that encodes a decoy receptor for TRAIL—a pro-apoptotic cytokine that is a promising target for the therapeutic inhibition of cancers. PARP13 destabilizes TRAILR4 mRNA post-transcriptionally in an exosome-dependent manner by binding to a region in its 30 untranslated region. As a consequence, PARP13 represses TRAILR4 expression and increases cell sensitivity to TRAIL-mediated apoptosis, acting as a key regulator of the cellular response to TRAIL. 1 Department of Biology, Massachusetts Institute of Technology, 500 Main Street, Cambridge, Massachusetts 02139, USA. 2 Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, Massachusetts 02139, USA. Correspondence and requests for materials should be addressed to P.C. (email: [email protected]). NATURE COMMUNICATIONS | 5:5362 | DOI: 10.1038/ncomms6362 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited.
    [Show full text]
  • Deletion of Irf4 in T Cells Suppressed Autoimmune Uveitis and Dysregulated Transcriptional Programs Linked to CD4+ T Cell Differentiation and Metabolism
    International Journal of Molecular Sciences Article Deletion of Irf4 in T Cells Suppressed Autoimmune Uveitis and Dysregulated Transcriptional Programs Linked to CD4+ T Cell Differentiation and Metabolism Minkyung Kang 1,†, Hyun-Su Lee 1,†, Jin Kyeong Choi 1,2, Cheng-Rong Yu 1 and Charles E. Egwuagu 1,* 1 Molecular Immunology Section, Laboratory of Immunology, National Eye Institute (NEI), National Institute of Health, Bethesda, MD 20892, USA; [email protected] (M.K.); [email protected] (H.-S.L.); [email protected] (J.K.C.); [email protected] (C.-R.Y.) 2 Department of Immunology, Jeonbuk National University Medical School, Jeonju, Jeonbuk 54907, Korea * Correspondence: [email protected]; Tel.: +301-496-0049; Fax: +301-480-3914 † These authors contributed equally. Abstract: Interferon regulatory factor-4 (IRF4) and IRF8 regulate differentiation, growth and functions of lymphoid and myeloid cells. Targeted deletion of irf8 in T cells (CD4-IRF8KO) has been shown to exacerbate colitis and experimental autoimmune uveitis (EAU), a mouse model of human uveitis. We therefore generated mice lacking irf4 in T cells (CD4-IRF4KO) and investigated whether expression of IRF4 by T cells is also required for regulating T cells that suppress autoimmune diseases. Surprisingly, we found that CD4-IRF4KO mice are resistant to EAU. Suppression of EAU derived in part from inhibiting pathogenic responses of Th17 cells while inducing expansion of regulatory lymphocytes that secrete IL-10 and/or IL-35 in the eye and peripheral lymphoid tissues. Furthermore, CD4- IRF4KO T cells exhibit alterations in cell metabolism and are defective in the expression of two Citation: Kang, M.; Lee, H.-S.; Choi, Ikaros zinc-finger (IKZF) transcription factors (Ikaros, Aiolos) that are required for lymphocyte J.K.; Yu, C.-R.; Egwuagu, C.E.
    [Show full text]
  • Znfs: Zinc Finger Families MBV4230 Zinc Finger Proteins
    ZNFs: zinc finger families MBV4230 Zinc finger proteins Zinc finger proteins were first discovered as transcription factors. Zinc finger proteins are among the most abundant proteins in eukaryotic genomes. Their functions are extraordinarily diverse include DNA recognition, RNA packaging, transcriptional activation, regulation of apoptosis, protein folding and assembly, and lipid binding. Zinc finger structures are as diverse as their functions. Odd S. Gabrielsen MBV4230 Zinc finger family: subfamilies with common DBD-structure TFIIIA - prototype and founder member Zinc fingers = Zn-structured domains binding DNA classical C2H2-fingers Nuclear receptors GATA-factors LIM domains GAL4-related factors Nucleocapsid proteins TFIIS RING finger PKC CRD Odd S. Gabrielsen MBV4230 Examples C4-type Zif C2H2-type Zif fra GATA-1 fra Sp1 (3.fngr) Zn++ LIM-domain type Zif PKC-type Zif fra ACRP Odd S. Gabrielsen MBV4230 Alignments C-C-H-H C-C-C-C Odd S. Gabrielsen The C2H2 subfamily MBV4230 Classical TFIIIA-related zinc fingers: n x [Zn-C2H2] History: Xenopus TFIIIA the first isolated and cloned eukaryotic TF Function: activation of 5S RNA transcription (RNAPIII) Rich source : accumulated in immature Xenopus oocyttes as “storage particles” = TFIIIA+5S RNA (≈ 15% of total soluble protein) Purified 1980, cloned in 1984 Mr= 38 600, 344 aa Primary structure TFIIIA Composed of repeats: 9x 30aa minidomains + 70aa unique region C-trm Each minidomain conserved pattern of 2Cys+2His Hypothesis: each minidomain structured around a coordinated zinc ion (senere bekreftet) ++ ++ ++ ++ Zn Zn Zn Zn Zn++ Zn++ Zn++ Zn++ Odd S. Gabrielsen MBV4230 Zinc finger proteins Finger-like i 2D Not in 3D Odd S.
    [Show full text]
  • The Heavy Metal-Regulatory Transcription Factor MTF-1
    Review articles Putting its fingers on stressful situations: the heavy metal-regulatory transcription factor MTF-1 P. Lichtlen** and W. Schaffner* Summary and the liver. They have the ability to bind heavy metals such It has been suggested that metallothioneins, discovered as zinc, cadmium, copper, nickel and cobalt (reviewed in about 45 years ago, play a central role in heavy metal metabolism and detoxification, and in the management of Ref. 7). Metallothioneins are involved in homeostatic regula- various forms of stress. The metal-regulatory transcrip- tion of zinc concentrations and also for the detoxification of tion factor-1 (MTF-1) was shown to be essential for basal non-essential heavy metals. As an example of the latter, and heavy metal-induced transcription of the stress- mammals store all cadmium taken up in food in a form tightly responsive metallothionein-I and metallothionein-II. Re- bound to metallothioneins, where it remains with a half-life of cently it has become obvious that MTF-1 has further roles (8) in the transcriptional regulation of genes induced by approximately 15 years in humans. The expression of the various stressors and might even contribute to some major metallothionein genes (MT-I and MT-II ) is induced at the aspects of malignant cell growth. Furthermore, MTF-1 is level of transcription by heavy metal load.(9) As shown by an essential gene, as mice null-mutant for MTF-1 die in Palmiter and colleagues, the promoter region of these metal- utero due to liver degeneration. We describe here the lothionein genes contain so-called metal responsive elements state of knowledge on the complex activation of MTF-1, and propose a model with MTF-1 as an interconnected (MREs) that can confer metal-inducibility to any reporter gene (10,11) cellular stress-sensor protein involved in heavy metal when placed in a promoter position or at a remote metabolism, hepatocyte differentiation and detoxifica- enhancer position.(12) Several laboratories, including ours, tion of toxic agents.
    [Show full text]
  • Structural Analyses of Zinc Finger Domains for Specific Interactions with DNA Ki Seong Eom1, Jin Sung Cheong2,3, and Seung Jae Lee3*
    J. Microbiol. Biotechnol. (2016), 26(12), 2019–2029 http://dx.doi.org/10.4014/jmb.1609.09021 Research Article Review jmb Structural Analyses of Zinc Finger Domains for Specific Interactions with DNA Ki Seong Eom1, Jin Sung Cheong2,3, and Seung Jae Lee3* 1Department of Neurosurgery, School of Medicine and Hospital, Wonkwang University, Iksan 54538, Republic of Korea 2Department of Neurology, School of Medicine and Hospital, Wonkwang University, Iksan 54538, Republic of Korea 3Department of Chemistry and Research Institute of Physic and Chemistry, Chonbuk National University, Jeonju 54896, Republic of Korea Received: September 19, 2016 Revised: September 26, 2016 Zinc finger proteins are among the most extensively applied metalloproteins in the field of Accepted: September 28, 2016 biotechnology owing to their unique structural and functional aspects as transcriptional and translational regulators. The classical zinc fingers are the largest family of zinc proteins and they provide critical roles in physiological systems from prokaryotes to eukaryotes. Two First published online cysteine and two histidine residues (Cys2His2) coordinate to the zinc ion for the structural October 6, 2016 functions to generate a ββα fold, and this secondary structure supports specific interactions *Corresponding author with their binding partners, including DNA, RNA, lipids, proteins, and small molecules. In Phone: +82-63-270-3412; this account, the structural similarity and differences of well-known Cys2His2-type zinc fingers Fax: +82-63-260-3407; such as zinc interaction factor 268 (ZIF268), transcription factor IIIA (TFIIIA), GAGA, and Ros E-mail: [email protected] will be explained. These proteins perform their specific roles in species from archaea to eukaryotes and they show significant structural similarity; however, their aligned amino acids present low sequence homology.
    [Show full text]
  • Mutations in the Glucocorticoid Receptor Zinc Finger Region That Distinguish Interdigitated DNA Binding and Transcriptional Enhancement Activities
    Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Mutations in the glucocorticoid receptor zinc finger region that distinguish interdigitated DNA binding and transcriptional enhancement activities Mark Schena^ Leonard P. Freedman, and Keith R. Yamamoto Department of Biochemistry and Biophysics, University at Cahfomia-San Francisco, San Francisco, California 94143-0448 USA Mammalian glucocorticoid receptors bind specifically to glucocorticoid response element (GRE) DNA sequences and enhance transcription from GRE-linked promoters in mammalian cells and in yeast. We randomly mutagenized a segment of the receptor encompassing sequences responsible for DNA-binding and transcriptional regulation and screened in yeast for receptor defects. The mutations all mapped to a 66-amino- acid subregion that includes two zinc fingers; in general parallel phenotypes were observed in yeast and animal cells. Mutants defective for DNA binding also failed either to enhance or to repress transcription. However, several mutations in the second finger selectively impaired enhancement; we suggest that such 'positive control' mutants may alter protein-protein contacts required for transcriptional activation. [Key Words: GRE; zinc finger domain; DNA binding; transcription] Received July 5, 1989; revised version accepted August 14, 1989. In the presence of bound hormone, the glucocorticoid trahedrally by four cysteine sulfur atoms, and that metal receptor protein regulates transcription initiation from coordination is essential for proper folding and DNA specific animal cell promoters used by RNA polymerase binding. In addition, a 28-amino-acid subfragment has II. The receptor enhances transcription by associating been identified, which lies downstream of the fingers, selectively with DNA sequences termed glucocorticoid that exhibits nuclear localization activity but fails to response elements (GREs) (Chandler et al.
    [Show full text]
  • Hedgehog Pathway-Regulated Gene Networks in Cerebellum Development and Tumorigenesis
    Hedgehog pathway-regulated gene networks in cerebellum development and tumorigenesis Eunice Y. Leea, Hongkai Jib, Zhengqing Ouyangc, Baiyu Zhoud, Wenxiu Mae, Steven A. Vokesf, Andrew P. McMahong, Wing H. Wongd, and Matthew P. Scotta,1 aDepartments of Developmental Biology, Genetics, and Bioengineering, Howard Hughes Medical Institute, and Departments of cBiology, dStatistics and Health Research and Policy, and eComputer Science, Stanford University, Stanford, CA 94305; bDepartment of Biostatistics, Johns Hopkins University, Baltimore, MD, 21205; fSection of Molecular Cell and Development Biology, Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712; and gDepartment of Molecular and Cellular Biology and Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138 Contributed by Matthew P. Scott, April 20, 2010 (sent for review November 24, 2009) Many genes initially identified for their roles in cell fate determi- genes influence murine MB development (18–21), so at least nation or signaling during development can have a significant part of the Shh-stimulated transcriptional program in GNPs is impact on tumorigenesis. In the developing cerebellum, Sonic active and functional during carcinogenesis. Genome-wide ex- hedgehog (Shh) stimulates the proliferation of granule neuron pression profiling has been widely used to understand the tran- precursor cells (GNPs) by activating the Gli transcription factors. scriptional effects of Shh signaling, but it does not distinguish Inappropriate activation of Shh target genes results in unrestrained direct and indirect effects. Many questions remain regarding the cell division and eventually medulloblastoma, the most common repertoire and regulation of Hh target genes and the extent of pediatric brain malignancy. We find dramatic differences in the their overlap in different tissues and cancers.
    [Show full text]
  • Regulation of NF-Κb Signaling by the A20 Deubiquitinase
    Cellular & Molecular Immunology (2012) 9, 123–130 ß 2012 CSI and USTC. All rights reserved 1672-7681/12 $32.00 www.nature.com/cmi REVIEW Regulation of NF-kB signaling by the A20 deubiquitinase Noula Shembade and Edward W Harhaj The NF-kB transcription factor is a central mediator of inflammatory and innate immune signaling pathways. Activation of NF-kBis achieved by K63-linked polyubiquitination of key signaling molecules which recruit kinase complexes that in turn activate the IkB kinase (IKK). Ubiquitination is a highly dynamic process and is balanced by deubiquitinases that cleave polyubiquitin chains and terminate downstream signaling events. The A20 deubiquitinase is a critical negative regulator of NF-kB and inflammation, since A20-deficient mice develop uncontrolled and spontaneous multi-organ inflammation. Furthermore, specific polymorphisms in the A20 genomic locus predispose humans to autoimmune disease. Recent studies also indicate that A20 is an important tumor suppressor that is inactivated in B-cell lymphomas. Therefore, targeting A20 may form the basis of novel therapies for autoimmune disease and lymphomas. Cellular & Molecular Immunology (2012) 9, 123–130; doi:10.1038/cmi.2011.59; published online 20 February 2012 Keywords: A20; inflammation; NF-kB; ubiquitin INTRODUCTION covalently attached to lysine residues on protein substrates and either The NF-kB transcription factor is ubiquitously expressed and is triggers proteasomal degradation, protein trafficking, signal transduc- activated by diverse immune stimulatory ligands, including inter- tion or the DNA damage response. Ubiquitination involves a three-step leukin-1b (IL-1b), tumor necrosis factor-a (TNF-a), bacterial lipo- enzymatic reaction catalyzed by three classes of proteins: E1 ubiquitin- polysaccharide (LPS) and antigenic peptides.
    [Show full text]